CTSS通过树突状细胞介导肠道免疫耐受失衡在肠易激综合征中的作用机制研究

The pathophysiology of IBS remains unknown. Recent evidence suggests that IBS pathology may result from a dysregulated immune response and breakdown of tolerance to the gut harmless antigens such as the commensal microbiota and food antigens. Dendritic cells are professional antigen presenting cells. Unlike other antigen presenting cells, DC have the unique ability to stimulate primary immune responses, and determine development of either immunogenic or tolerogenic immune responses. It is clear that intestinal DC play a crucial role in mediating mucosal tolerance against commensal microbiota and food antigens, preventing unnecessary inflammation and hypersensitivity. Dysregulation of tolerogenic properties in intestinal DC may lead to IBS. However, its mechanisms remain poorly understood. In previous study, we found that in IBS rats, the number of colonic DC increased, and function of mesenteric lymph node(MLN)DC changed, co-cultured MLN DC with CD4+T cells showed a low level of IL-10 secretion, which indicated that the phenotype and function of DC changed, contributing to the breakdown of homeostasis of intestinal mucosal immune tolerance. It finally resulted in the onset of IBS. Furthermore, we identified differentially expressed proteins in colon tissue of IBS rats compared with healthy controls by 2-D gel electrophoresis, MALDI-TOF-MS. Our results showed CTSS was up-regulated in colon mucosa of IBS rats, while it was closely related to antigen presentation and the regulation of DC maturation. Thus, we suppose CTSS may influence functional status of intestinal DC, causing an imbalance of intestinal immune tolerance, which would be involved in the pathogenesis of IBS. This program intends to study further on the phenotype and the functional states of primary intestinal dendritic cells in IBS rats, and construct a recombinant lentiviral vector carrying CTSS gene and a recombinant short hairpin RNA (shRNA) lentiviral vector carrying CTSS gene, establish the stably transfected DC, study the phenotypic and functional changes of intestinal DC, and intraperitoneal injection of the stably transfected DC in rats, observe IBS related features in rats. Therefore, it might help to reveal the effect of CTSS on the imbalance of intestinal immune tolerance in IBS rats, mediated by DC. And it would provide new insights for the pathophysiology of IBS and clinical treatment.

肠道免疫系统对无害抗原的免疫耐受失衡是肠易激综合征(IBS)发病的重要机制，树突状细胞（DC）在维持肠道免疫系统动态平衡中发挥重要作用。我们前期发现IBS大鼠肠道DC抑制T细胞分泌IL-10，IL-10与免疫耐受密切相关，推测IBS大鼠肠道DC的功能状态发生变化；蛋白质组学研究发现与抗原提呈相关的半胱氨酸组织蛋白酶S（CTSS）在IBS肠道中表达增高，且IBS大鼠肠道DC的CTSS表达上调，提示CTSS可能通过影响DC的功能状态，导致肠道免疫耐受失衡，参与IBS的发病。本项目拟在先前基础上进一步明确IBS肠道DC的表型及功能状态；构建CTSS慢病毒载体，建立CTSS过表达和shRNA稳转DC细胞株，观察CTSS对肠道DC表型及功能的影响；探讨CTSS稳转DC对大鼠IBS相关特征的影响。本项目的实施有助于揭示CTSS在DC介导的IBS肠道免疫耐受失衡中的作用，为IBS的诊治提供候选靶点。

肠道免疫系统对无害抗原的免疫耐受失衡是肠易激综合征(IBS)发病的重要机制，树突状细胞（DC）的功能状态决定了特异性免疫细胞的分化方向和反应强度，在激发免疫应答和诱导免疫耐受中发挥着关键作用，是维持肠道免疫系统动态平衡的中心环节。蛋白半胱氨酸组织蛋白酶S（CTSS）主要表达于抗原提呈细胞：如DC，参与MHC-Ⅱ类分子调节的抗原提呈作用。本项目以IBS差异蛋白-CTSS为研究切入点，初步阐明CTSS对DC表型及功能的影响，从而引起肠道免疫耐受失衡，导致IBS发病的机制。本研究创新性发现，IBS动物模型肠道DC的CTSS蛋白表达水平明显上调，并且IBS动物模型肠道DC表面MHC-II类分子表达升高，而表面分子MHC-I类分子和共刺激分子CD80、CD86表达降低；DC与T淋巴细胞共培养上清中IL-10、Foxp3水平降低，IL-12水平升高；DC的Foxp3、TGF-β1和β-catenin的蛋白表达减少，提示IBS动物模型肠道DC的表型及功能状态发生变化，促进T细胞增殖分化，激发肠道异常的免疫应答，诱导肠道免疫耐受失衡。采用基因沉默技术，将CTSS基因干扰慢病毒转染DC，发现DC刺激初始T细胞增殖的能力减弱。对IBS大鼠进行CTSS基因干扰慢病毒干预，引起大鼠内脏敏感性的下降，DC表面高表达MHC-I类分子，而MHC-II类分子表达相对较低。提示CTSS通过调控DC的表型和功能状态，促进肠道免疫应答的异常激活，导致肠道免疫耐受失衡，从而参与了IBS的发病。研究结果有望为IBS发病机理提供新思路，为IBS的诊治提供候选靶点。